Pentacyclic triterpenes

ABSTRACT

The present invention relates to fuingicidally effective compositions containing at least one pentacyclic triterpene compound.

This application is a divisional of copending U.S. application Ser. No.09/207,406 filed on Dec. 8, 1998 the disclosure of which is hereinincorporated by reference.

FIELD OF THE INVENTION

The invention relates to plant protection compositions includingpentacyclic triterpenes and methods for use thereof.

BACKGROUND

Outer layers of plants such as leave cuticle, fruit peels, as well asbark protect the plant against abrasion, prevent water loss, and alsoprotect against pathogenic microorganisms. The breaking through theplant cuticle is a prerequisite for a pathogen to be able to enter theplant's internal tissue.

The mechanism, by which plants naturally defend themselves against thisearly stage of pathogenesis has not been fully understood. The initialprocess of fungal propagules attaching to a host plant is essential tothe successful establishment of pathogenesis. The established facts thataerial fungal pathogens bind strongly to very hydrophobic surfacessuggests that hydrophobic forces are involved in the attachmentprocesses.

The attachment of aerial fungal pathogens involves an active process ofsecretion of extracellular mucilages or adhesives, which may startwithin minutes after contact with the host. Other reported components inthe adhesive secretions include enzymes, among them esterases andcutinases. The erosion of cuticular waxes adjacent to and underlying theconidium may became observable within 20 min of liquid release. Thegrowth of the appressorial germ tube appears to be limited to the zoneof deposition of the liquid film.

Some studies have suggested that preparation of the infection courtinvolves active dissolution of the host cuticle by foliar pathogen. Itis also believed that this dissolution is the purpose of these enzymes.Nicholson, R. L., and L. In: The Fungal Spore and Disease Initiation inPlants and Animals., Eds. Cole, G. T., and Hoch, H. C.,1991 PlenumPress, New York, 3-23. Thus, the cuticle has to be penetrated by theattacking pathogen before the sequential steps of disease developmentoccur. Some fungal spores help themselves with mechanical force exertedby the infection structure in addition to the enzymatic degradation.(Köller, W. in: The Fungal Spore and Disease Initiation in Plants andAnimals., Eds. Cole, G. T., and Hoch, H. C., 1991 Plenum Press, NewYork, 219-246.

Pentacyclic triterpenes (PT) are among the most common plant secondarymetabolites, but their function in plants have not been understood. Theyare usually concentrated in the outermost layers such as plant cuticle,fruit peel and bark. In some cases, these layers contain very highconcentration of pentacyclic triterpenes. For example, an apple peelcontains about 0.1 grams of ursolic acid per fruit, and the outer barkof white birch species contains up to 40% w/w of betulin. The amount ofbetulin obtainable from the birch bark waste in the wood-workingindustry in Finland is estimated at about 150,000 tons per annum. Atpresent, waste bark is used as a low value fuel for energy production.Jääskeläinen, P. (1981) Pap. Puu 63, 599-603.

Literature supplies numerous examples of enzymes that can be inhibitedby PT, indicating the ability of PT to act broadly in a non-specificmode on multiple targets. See for example, (a.) Büchler et al. (1991)Biochem. Biophys. Acta 1075, 206-212, Inhibition of rat renal11β-hydroxysteroid dehydrogenase by steroidal compounds andtriterpenoids; structure/function relationship; (b.) Koch et al. (1994)Phytother. Res. 8, 109-111, In vitro inhibition of adenosine deaminaseby a group of steroid and triterpenoid compounds.; (c.) Najid et al.(1992) FEBS 299, 213-217, Characterization of ursolic acid as alipoxygenase and cyclooxygenase inhibitor using macrophages, plateletsand differentiated HL60 leukemic cells.; (d.) Pengsuparp et al. (1994)J. Nat. Prod. 57, 415-418, Pentacyclic triterpenes derived fromMaprounea africana are potent inhibitors of HIV-1 reversetranscriptase.; (e.) Simon et al. (1992) Biochem. Biophys. Acta 1125,68-72, Inhibition of lipoxygenase activity and HL60 leukemic cellproliferation by ursolic acid isolated from heather flowers (Callunavulgaris).; (f.) Ying et al. (1991) Biochem. J. 277, 521-526 Inhibitionof human leucocyte elastase by ursolic acid. Evidence for a binding sitefor pentacyclic triterpenes. The disclosures of each of these referencesis herein incorporated by reference.

In plant tissue cultures, stress induced by inactivated fungi or fungalenzymes has been used to enhance production of biologically activesecondary metabolites. In several instances it has been reported thatthis fungal elicitation led to overproduction of pentacyclic triterpenesinstead of some other expected metabolites. Suitable example is given byVan der Heijden et al., (1988) Plant Cell Rep. 7, 51-54, where tissuecultures of Tabernaemontana spp., normally producing indole alkaloids,were subjected to stress induced by either fungi, bacteria, or enzymecellulase or pectinase. When stressed, however, the culture produced upto 3.3 times the normal rate of the ursane-type pentacyclic triterpenes(2% of dry mass) but no increase in the production of indole alkaloidsoccurred.

Other experiments with Tabernaemontana divaricata treated with Candidaalbicans elicitor led to production of a series of pentacyclictriterpenes of the ursane and oleane types, and was accompanied byinhibition of both growth and indole alkaloid accumulation (Van derHeijden et al., (1989) Phytochemistry 28, 2981-1988).

Also, the tissue culture of Tripterygium wilfordii, normally a source ofpotent cytotoxic diterpenes, stressed by fungal Botrytis elicitordramatically enhanced production of oleane-type pentacyclic triterpenesbut not the diterpenes, prompting a conclusion that only triterpenes areinducible anti-microbial phytoalexins. Kutney, et al., (1993)Anti-inflammatory oleane triterpenes from Tripterygium wilfordii cellsuspension cultures by fungal elicitation. Plant Cell Rep. 12, 356-359.

The conventional treatments for leafy and grassy plants that have beenattacked by fungi and bacteria are usually exercised after an outbreakoccurs. The affected plants are then treated with one or more of thecommercial synthetic contact antimicrobial sprays at application ratesthat do not pose phytotoxicity concerns. Effective treatment rates mustbe balanced against the risks of harming the treated plant withchemicals that are structurally unrelated to those in the plantphysiology. It would be desirable to have a protective agent that iseffective against microbial pathogens which would also work in a manneranalogous to the plant's natural defense mechanisms to reduce the riskof phytotoxicity. One of the problems associated with treatinghydrophobic leaf surfaces is an effective application of the material.Current spraying techniques result in a portion of the sprayed materialfalling to the ground directly or after being washed off from rainoccurring shortly after application. Either event increases concerns forenvironmental contamination.

Protective agents that are applied by spraying should remain on theplant surface for a time sufficient to serve their intended function.Stability against ultraviolet and visible light is, therefore, a concernfor foliar treatments. It would be desirable to develop fungicides forfoliar application that resist degradation by exposure to ultraviolet aswell as visible light.

Moreover, invading organisms have been known to evade the effects of atreatment agent by mutation and propagation of resistant strains. Suchdeveloped resistance is economically detrimental because it forces thediscovery of new treatments. It would be helpful to provide plantanti-infective agents which cannot be evaded by mutating pathogens.

SUMMARY OF THE INVENTION

It is an object of the invention to provide a composition and processfor use thereof for protecting plant surfaces against microbialpathogens with ingredients that are compatible with natural plantdefense mechanisms.

It is another object of the invention to provide a composition andmethod of use that poses little risk to the environment, humans, orbeneficial insects.

It is another object of the invention to provide plant anti-infectiveagents, which cannot be evaded by developing resistance by mutatingpathogens.

It is yet another object of the invention to provide a compositionhaving good resistance to degradation by ultraviolet exposure, longstorage times, and good inherent sticking ability to plant surfaces.

In accordance with these and other objects of the invention, which willbecome apparent from the description herein, a composition according tothe invention comprises: a pentacyclic triterpene or a mixture ofpentacyclic triterpenes, including some plant extracts that areparticularly rich in pentacyclic triterpenes, having as main componentscompounds described by any of formulas I, II, or III.

A method according to the invention comprises applying to plant surfacesan effective amount of a composition comprising a pentacyclic triterpeneor a mixture of pentacyclic triterpenes exhibiting any of formulas I, IIor III.

Formulas I, II, and III are:

wherein:

R¹=Me, CH₂OH, CH₂OY¹, CH₂O—X—OH, CH₂O—X—OY¹, CH₂O—X—Y², CH₂O—X—Y³,CH₂NHY¹, CH₂NY¹ ₂,CH₂Y³, CH₂NH—X—OH, CH₂NH—X—Y², CH₂NH—X—Y³,CH₂NH—X—OY¹, CH₂OC(O)—OY¹, CH₂O—X—OY¹, CO₂Y¹, COY³, COY², CHO,CH═N(CH₂)_(m)(O(CH₂)_(m))_(n)R⁴, or CH═N(CH₂)_(m)(O(CH₂)_(m))_(n)Y²;

R², R³=H, OH, OY¹, O—X—OH, O—X—OY¹, O—X—Y², Y³, NHY¹, NY¹ ₂, Y³,NH—X—OH, NH—X—Y², NH—X—Y³, NH—X—OY¹, NY¹—X—OH, NY¹—X—Y², NY¹—X—Y³, orNY¹—X—OY¹; provided that one of R² and R³ is H or that R² and R³together denote carbonyl oxygen;

R⁴=H, OH, OY¹, or Y³;

Y¹=H, alkyl of 1-30 carbon atoms, straight chain or branched, cycloalkylof 3-30 carbon atoms, alkanyl of 3-30 carbon atoms, oxyalkyl of 4-30carbon atoms, phenylalkyl of 7-30 carbon atoms, or phenoxyalkyl of 7-30carbon atoms;

Y²=NH₂, NHY¹, or NY¹ ₂;

Y³=—(O(CH₂)_(m))_(n)R⁴ or —(O(CH₂)_(m))_(n)Y², where m=2-4 and n=1-230;

X=—OC(CH₂)_(p)CO— where p=1-22.

The present invention provides a composition and method of use that areparticularly effective in preventing outbreaks of airborne fungal andbacterial diseases on treated plant surfaces. The pentacyclictriterpenes of the composition are applied to form a film over the plantsurface. The film of material prevents the prerequisite attachment ofaerial fungal pathogen and penetration of the plant cuticle. Inaddition, the compounds are not phytotoxic and are compatible withnatural plant defenses

DETAILED DESCRIPTION

The present invention relates to compositions containing and methods ofusing pentacyclic triterpenes having the following formulas I, II, orIII:

wherein:

R¹=Me, CH₂OH, CH₂OY¹, CH₂O—X—OH, CH₂O—X—OY¹, CH₂O—X—Y², CH₂O—X—Y³,CH₂NHY¹, CH₂NY¹ ₂,CH₂Y³, CH₂NH—X—OH, CH₂NH—X—Y², CH₂NH—X—Y³,CH₂NH—X—OY¹, CH₂OC(O)—OY¹, CH₂O—X—OY¹, CO₂Y¹, COY³, COY², CHO,CH═N(CH₂)_(m)(O(CH₂)_(m))_(n)R⁴, or CH═N(CH₂)_(m)(O(CH₂)_(m))_(n)Y²;

R², R³=H, OH, OY¹, O—X—OH, O—X—OY¹, O—X—Y², Y³, NHY¹, NY¹ ₂, Y³,NH—X—OH, NH—X—Y², NH—X—Y³, NH—X—OY¹, NY¹—X—OH, NY¹—X—Y², NY¹—X—Y³, orNY¹—X—OY¹; provided that one of R² and R³ is H or that R² and R³together denote carbonyl oxygen;

R⁴=H, OH, OY¹, or Y³;

Y¹=H, alkyl of 1-30 carbon atoms, straight chain or branched, cycloalkylof 3-30 carbon atoms, alkanyl of 3-30 carbon atoms, oxyalkyl of 4-30carbon atoms, phenylalkyl of 7-30 carbon atoms, or phenoxyalkyl of 7-30carbon atoms;

Y²=NH₂, NHY¹, or NY¹ ₂;

Y³=—(O(CH₂)_(m))_(n)R⁴ or —(O(CH₂)_(m))_(n)Y², where m=2-4 and n=1-230;

X=—OC(CH₂)_(p)CO— where p=1-22.

The preferred pentacyclic triterpenes include betulin, betulinic acid,ursolic acid, oleanolic acid, betulin mono- and di-succinate orglutarate, as well as polyethylene glycol derivatives of thereof.

Particularly useful are those pentacyclic triterpenes exhibiting an IC₅₀value against human leucocyte elastase at concentration less than about15 micromolar (μM), more preferably an IC₅₀ value of less than about 10,and most preferably an IC₅₀ value at less than about 8 micromolar. TheIC₅₀ value represents the concentration of an inhibitor, which can beexpressed in micromoles per liter, at which activity of an enzyme isreduced by 50%. Thus, lower IC₅₀ values suggest higher levels of enzymeinhibitory activity.

The pentacyclic triterpenes of the invention are the same as, derivedfrom, synthesized, or otherwise related to those found naturally in theouter surfaces of plants: leaves, fruits, bark, and are subjected topathogenesis involving enzymatic degradation of cuticle. For the presentinvention, the pentacyclic triterpenes or their derivatives are appliedto the exposed outer plant surfaces in conjunction with a suitablecarrier such as water, an aqueous film-forming solution, detergents,emulsion forming additives, suitable polymers to enhance physicalproperties of the sprayed layer.

Pentacyclic triterpenes can be obtained by extracting the pentacyclictriterpene-containing plant tissues with one or more organic solventssuitable for the triterpenes. Preferred plant tissue sources for PTinclude bark from white birch trees, apple peels, and the leaves ofplants belonging to Vaccinium and Myristica spp. Useful solvents for theextraction include ethyl acetate, acetone, methyl ethyl ketone, ethanol,propanol, isopropanol, methanol, methylene chloride, chloroform, ortheir mixtures.

The pentacyclic triterpenes or their polyethylene glycol derivatives ofthe invention should be formed in a non-crystalline state into a wellmixed colloidal suspension for application as a uniform coating on thetreated plant surfaces. The uniform coating helps to ensure that plantsurfaces are well protected against pathogens with the exception ofunder surfaces or secluded regions unable to be reached by conventionalspraying equipment for liquid formulations.

The pentacyclic triterpene compounds of the invention are, however,crystalline solids of hydrophobic character. The solids can be dissolvedin a number of solvents suitable for agricultural use. If desired, thesolutions or colloidal concentrates of pentacyclic triterpenes can beprepared for shipping and storing. This concentrate can then be furtherdiluted for use by a formulator or applicator.

A preferred solvent for pentacyclic triterpene solids contains about1-25 wt % acetone, about 0-10 wt % dimethylsulfoxide (DMSO), 0-35%polyethyleneglycol ester of an aliphatic acid, and about 0-25 wt % of asurfactant such as commercially available detergents like Tween 80™ orPalmolive™ dishwashing detergent. Generally, this solvent mixture maycarry a concentration of a PT being 1500-4000%, by weight, of thatneeded for application to the plants.

The concentrate can be further diluted with 100-4000%, preferably300-1000% by weight of water to make a sprayable composition accordingto the invention. Particularly useful concentrations are within therange from about 7-30 grams per gallon of water. Adequate mixingrequires only low to moderate shearing to ensure adequate mixing of theconcentrate during dilution. For example, metering the concentrate intoa reservoir attached to a venturi mixer would provide adequate shear tocompletely mix the concentrate with additional water.

Pentacyclic triterpenes or their polyethylene glycol derivativesaccording to the invention are applied at a rate sufficient to preventpathogenic infections. The inhibitory properties of PT are utilized bythe plants to inactivate the enzymes excreted by the fungal spore inorder to degrade the plant cuticle. Suitable application rates foreffective protection include rates within the range from about 0.1-1000kg/h. Preferably, the application rate is within the range from about0.1-100 kg/h. The specific application rate that is best for aparticular type of plant in a particular region is readily determined bythe application of the ordinary skill in the art. The applicationshould, however, be designed to fall on and cover the exposed leafsurfaces of the plants being treated such as it occurs with conventionalfoliar treatments using conventional foliar spraying equipment.

If desired, the PT or their polyethylene glycol derivatives may beapplied in conjunction with one or more inert or active ingredients.Exemplary materials include dyes, additives affecting stability of theconcentrate and additives affecting physical properties of the sprayedlayer, foliar fertilizers, fungicides, and insecticides.

Virtually any plant that may get infected through the waxy cuticle layercan be beneficially treated with compositions according to the presentinvention. Commercial plants that would benefit include grain grasses(e.g., rye, wheat, and barley), tomato, bean, pepper, wheat, and peanutplants.

Grain grasses may benefit in particular from the present invention.These plants do not generally produce sufficient levels of pentacyclictriterpenes, if at all, to inhibit enzymatic attacks by invadingmicroorganisms. Natural pathogens of these plants are generally notadapted to produce sufficient amounts of enzymes to overcome theinhibiting effects of the externally applied pentacyclic triterpenecompounds.

Plants susceptible to small insect (e.g., aphids) infestation alsobenefit from treatment according to the invention. Ursolic acid showedtoxicity and feeding deterrent effects towards the mites and theirsurvival. The mites' reproductive indexes decreased in direct proportionto ursolic acid content in the diet, and in addition, ingestion time ondiet containing ursolic acid was reduced about 30% (Varanda et al.,1992). These observations imply a possibility that the insect'sdigestive enzymes were compromised by ursolic acid.

The mode of inhibition of enzymes by PT is non-specific and is basedprimarily on hydrophobic interaction with an enzyme's hydrophobicdomain. This property suggests that the likelihood for developingmicrobial pathogen resistance through mutation is remote. Pathogens hadto deal with the presence of PT in the plant cuticle for as long as theplants existed and it could be assumed that microbial resistance to thePT has been already optimized. The PT based formulations for plantprotection should be advantageous against the pathogenic microorganismsthat use enzymes to break through the cuticle, such as fungi, bacteria,nematodes and viruses. These formulations should be also advantageousagainst small insects, acting inhibitory on their digestive enzymes. Bycovering the leafy surface with a film containing pentacyclictriterpenes, the passive defensive properties of the cuticle areenhanced, which decreases or entirely prevents successful pathogenicpenetration.

The following examples are included to assist in an understanding of theinvention and are not intended to limit the scope of the attachedclaims.

EXAMPLES

Commercially available compounds were acquired either fromExtrasyntheses (France), Aldrich, Sigma or ICN Pharmaceuticals. Meltingpoints were obtained using a Reichert Thermovar melting point apparatusand are corrected. Hnmr spectra were acquired in C₅D₅N with TMS as aninternal standard (δ=0.0 ppm) using a Bruker WM-250 or 270 NMRspectrometer. Mass spectra were acquired using a Finnigan 4023 EI/CIGCMS mass spectrometer with Super-INCOS software and direct insertionprobe.

Examples 1a and 1b Synthesis of 28-hemisuccinylbetulin and3,28-Di-hemisuccinylbetulin. Example 1a

A reaction mixture is prepared by dissolving imidazole (4 eq) andsuccinic anhydride (1.08 eq) in N-methylpyrrolidone. The volume of thesolvent is about 4.5 times the weight of betulin. When the two reagentsare dissolved, betulin (1 eq) is added. The reaction mixture is left atroom temperature with occasional shaking. After approximately 48 hrs,about 92-95% of the betulin has been converted into28-hemisuccinilbetulin, with 2-4% as unconverted betulin anddiacylbetulin.

The 28-hemisuccinilbetulin is purified by pouring the reaction mixtureinto a 20× volume of water with slow stirring. The water is decantedfrom the solid residue and the washing is repeated with several newportions of water. The solid is separated, dried and dissolved in amixture of chloroform and isopropanol. The chloroform is removed byevaporation enabling 28-hemisuccinylbetulin to form and crystallize. The28-hemisuccinylbetulin crystals are white with a melting point of242-244° C.

Calculations for C₃₄H₅₄O₅, C, 75.22%, H, 10.03; found C, 75.0, H, 9.91.The ¹Hnmr (DMSO-d₆) gave peaks at: 4.86 and 4.72 (1H, s, each, CH₂═C),4.57 and 4.14 (AB pattern, J=12 Hz, OCH₂O), 3.44 (1H, t, J=8.6 Hz), 2.95(4H, s, CHOCH₂CH₂CO), 1.71, 1.22, 1.02, 0.98, 0.98, 0.84 (s, each, Me);eims (m/z, rel. int.): 524 (20, [M−H₂O]+), 424 (15), 411 (27), 207 (38),203 (41), 189 (100), 135 (70), 119 (61), 107 (62), 95 (81), 81 (79),55(90%); cims: 543 (11, M+), 525(64, [MH−H₂O]+), 425(80), 407 (100), 217(91), 203 (90), 191 (48), and 189 (52%).

Example 1b

Betulin, 443 mg (1 mmole), imidazole, 227 mg (4 mmoles), succinylanhydride, 400 mg (4 mmoles) and 0.6 ml of methylpyrrolidone werecombined in a vial. The reaction mixture was maintained at 70° C. for 20hrs. The reaction mixture was then added dropwise to 100 ml of water andstirred for half an hour. The resulting precipitate was filtered off,re-suspended and agitated in another portion of water in order to removethe remaining reactants. This procedure was repeated several times untilno more of the reactants were washed out. The solid was collected, driedand dissolved in acetone. The acetone solution was filtered andevaporated to give 470 mg of 3,28-dihemisuccinylbetulin.

Calculated for: C₃₈H₅₈O₈·¼H₂O, MW 642.84; C, 70.5%, H, 9.1%; found C70.43%, H 9.15%. 1H NMR: 4.70 and 4.61 (1H, s, each, ═CH2), 4.52 (1H,dd, J₁=5.4 Hz, J₂=10.0 Hz), 4.33 and 3.90 (1H, d, each, AB pattern,J=11.1 Hz, OCH₂), 2.63 -2.71 (8H, m, succinyl chain), 1.70, 1.05, 0.99,0.87, 0.86, and 0.85 (Me, s, each). MALDI-TOF-MS gave m/z 665 (M+Na⁺)and also m/z 542 and 525.

Example 2 Synthesis of Acetylbetulinic Acid.

Betulinic acid, 100 mg (0.22 mmoles) was added to a solution containing4 mg of 4-dimethylaminopyridine (DMAP) and 0.5 ml of (CH₃CO)₂O in 5 mlof CH₂Cl₂. After 2 hrs CH₂Cl₂ was removed in vacuo, and the remainingresidue stirred with 25 ml of water. During the stirring enough of K₂CO₃was added to decompose an excess of acetic anhydride, after which thereaction mixture was extracted with 25 ml of CH₂Cl₂. The extract wasevaporated to dryness and the resulting solid crystallized from MeOH togive 104 mg of acetylbetulinic acid as white crystals, m.p. 285° C.

Calculations for C₃₂H₅₀O₄, C, 77.06, H, 10.10; found C, 77.02, H,10.08%. The ¹Hnmr DMSO-d₆) gave peaks at: 4.93 and 4.76 (1H, s, each,CH₂═C), 4.67 (1H, dd, C3—H, J=5.0, 10.0 Hz), 2.05 (s, CH₃CO), 1.78,1.06, 1.00, 0.87, 0.84 and 0.73 (each, s, CH₃); eims (m/z, relativeintensity): 499 (1, MH+), 452 (2), 438 (38), 395 (26), 189 (40), 43(100%); cims (m/z, rel. intensity): 499 (37, MH+), 440 (27), 439 (100),437 (18), 393 (20), 203 (15), 191 (24%).

Example 3 Synthesis of 3-methanesulfonylbetulinic Acid.

Betulinic acid, 100 mg (0.22 mM) was dissolved in 5 ml of pyridine andtreated with 100 mg (0.88 mmoles) of CH₃SO₂Cl. After 16 hrs, thepyridine was removed in vacuo and the resulting residue suspended in 10ml of water. Excess of MeSO₂Cl was decomposed with an aqueous NaHCO₃solution, and the reaction mixture extracted with 10 ml of CH₂Cl₂.Evaporation of the solvent gave a crude product containing3-methanesulfonylbetulinic acid, which was purified by columnchromatography over 3 g of silica gel using CH₂Cl₂ with 0 to 5% gradientof MeOH. The chromatography afforded 3-methanesulfonylbetulinic acid,which on crystallization from MeOH gave 62 mg of white crystals, m.p.210-212° C.

Calculations for C₃₁H₅₀O₅S: C, 69.62, H, 9.42, S, 6.00; found C, 69.78,H, 9.46, S, 5.90%. ¹Hnmr (DMSO-d₆): 4.95 and 4.78 (1H, each, s, CH₂═C),4.50 (1H, dd, J 4.6, 11.8 Hz), 3.30 (3H, s, CH₃SO₂), 1.79, 1.07, 1.06,1.00, 0.82, 0.72 (each, s, CH₃); eims (m/z, relative intensity): 438(9), 423 (11), 395 (72), 259 (12), 161 (26), 135 (62), 121 (100), 107(60), 93 (61), 79 (54%); cims (m/z, rel. intensity): 535 (11, MH+), 439(91), 423 (25), 395 (52), 249 (12), 203 (28), 191 (34), 97 (100%).

Example 4 Synthesis of the Methyl Ester of 3-methanesulfonylbetulinicAcid.

Betulinic acid methyl ester, 500 mg (1.06 mmoles), and 320 mg (3.3mmoles) of Me₃N were dissolved in 10 ml of CH₂Cl₂. The solution wascooled in an ice-bath and treated with 190 mg (1.7 mmoles) of MeSO₂Cl.The reaction mixture was allowed then to warm up to room temperature andleft overnight. Evaporation of the solvent in vacuo left a residue,which was dissolved in 10 ml of Et₂O and washed 3 times with 5 ml H₂O.The etheral layer was dried over MgSO₄, filtered, and evaporated to give582 mg of the methyl ester of 3-methanesulfonylbetulinic acid.Recrystallization from EtOH gave crystals with a melting point of 190°C.

Calculation for C₃₂H₅₂O₅S: C, 70.03, H, 9.55, S, 5.84; found C, 69.85,H, 9.50, S, 5.65%. ¹Hnmr (DMSO-d₆): 4.89 and 4.73 (1H, each, CH₂═C),4.47 (1H, dd, C3—H, J=2.5, 12.5 Hz), 3.70 (s, OCH₃), 3.29 (s, CH₃SO₂),1.72, 1.04, 0.99, 0.92, 0.81, 0.72 (s, each, CH₃); eims (m/z, relativeintensity): 452 (9), 409 (14), 341 (8), 273 (15), 255 (12), 189 (100),175 (47), 121 (72), 107 (72), 93 (76), 79 (85%); cims (m/z, rel.intensity): 549 (16%, MH+), 453 (100), 393 (19), 203 (13), 189 (12), 97(80%).

Examples 5-24 Elastase Inhibition

Studies, performed with human leucocyte elastase (HLE) indicated thatmany common naturally occurring PTs with lupane, oleane, and ursaneskeletons inhibit HLE at low micromolar concentrations. Severalderivatives of these PTs were prepared using synthetic methodology toexplore structure-activity relationship and on testing they alsoinhibited HLE. The values of inhibitory constants, IC₅₀'s obtained fromHLE inhibition, for the tested compounds, are given in Table 1.

Stock solutions were mixed to yield a reaction mixture consisting of 0.5mM MeO-Suc-Ala-Ala-Pro-Val-pNA (Sigma) and 0.7 μmg/ml human leucocyteelastase (Elastin Products) in 0.1 M Tris, 0.5 M NaCl and 3% DMSO (v/v)at a pH of 7.5. Test compounds were solubilized in DMSO. Equal volumesof enzyme and inhibitor were mixed and allowed to incubate for 15minutes at room temperature. The reaction was started by the addition ofa third equal volume containing substrate. Reactions were carried out inmicroliter plates. Appearance of p-nitroaniline was monitored at 405 nmin a Dynatech microelisa Reader M600. Initial rates in the presence andabsence of test compound were compared to determine IC₅₀ values whichare reported in Table 1.

TABLE 1 Ex. Compound Name IC₅₀ (μM) 5 Lupeol 8.4 6 Lupeol acetate 14.9 7Betulin 5.0 8 28-Succinylbetulin 6.3 9 Betulinic acid 3.3 10 Methylester of betulinic acid 5.7 11 Acetylbetulinic acid 7.0 123-Ketobetulinic acid 11.0 13 Methanesulfonylbetulinic acid 11.2 14Methyl ester of 3-methanesulfonylbetulinic acid 7.3 15 β-Amyrin 5.4 16Uvaol 4.5 17 Ursolic acid 7.7 18 Ursolic acid, Me ester 4.9 19 α-Amyrin21.1 20 Oleanolic acid 18.7 21 Oleanolic acid, Me ester 8.5 22Echinocystic acid 21.1 23 Hederagenin 16.8 24 Caulophyllogenin 0% @ 21°

Further experiments, carried out with ursolic acid and oleanolic acid,indicated that these compounds can inhibit also other enzymes, such asplasmin and urokinase. The values of inhibition, measured at 22 μM inthe plasmin assay were 43% for ursolic acid and 25% for oleanolic acid,while in the urokinase assay were 83% for ursolic acid and 40% foroleanolic acid. For betulinic acid, in the following assays, IC₅₀ valueswere determined to be: thrombin 2.3, trypsin 8.5, plasmin 6.2, andurokinase 5.0 μM. The apparent lack of specificity within the testedenzymes and an observation that the inhibition ceases in the presence of0.1% of albumin suggested that the inhibition results from anon-specific binding to proteins. Thus, PTs appear to bindnon-covalently to hydrophobic domains of enzymes and either block anaccess to the enzyme active site or cause conformational change, bothresulting in the enzyme's inability to perform its function.

The most potent inhibitors of the investigated group of PTs contain oneto three oxygenated substituents, such as hydroxy or carboxy groups.These moieties contribute to the overall inhibitory effect of thecompounds.

Examples 25 Fungicidal Activity

Tomato, bean, pepper, wheat, and peanut plants are grown for one tothree weeks (depending upon species) in the greenhouse. Two pots, whichrepresent two replicates, of each plant species are placed into a flatsuch that each flat contains all the plants to be sprayed by onecompound. The plants in each flat are sprayed to runoff with the desiredspraying solution or with fungicide standard. As a control, check plantsare sprayed with water. The plants are allowed to air-dry two to threehours. After the plants are dry, they are sorted and grouped by plantspecies.

Table 2 identifies the compositions of examples 25-28 that were testedfor their ability to protect hydrophobic plant surfaces:

TABLE 2 Example Material 25 Control vehicle containing acetone 18 ml,dimethylsulfoxide 4 ml, and Palmolive ™ detergent 200 mg. Tested as 3%aqueous concentration. 26 40 mg betulin dissolved in 1.5 ml of vehicleand added to 98.5 ml of water. 27 80 mg betulin, 3.0 ml of vehicle, and97 ml of water. 28 40 mg of ursolic acid, 2.0 ml of vehicle, and 98 mlof water.

The plant pathogenic fungi Phytophthora infestans (Pi), Alternariasolani (As), Botrytis cinerea (Bc), and Cercospora arachidicola (Ca)were grown in the laboratory on appropriate media. Inoculum from eachfungus was harvested and concentrations adjusted to predeterminedlevels. The obligate plant pathogenic fungi (Erysiphe graminis f.sp.tritici, Puccinia recondita f.sp. tritici) are harvested from theirhosts in the greenhouse and concentration are adjusted to predeterminedlevels.

The plants previously treated with test compounds are sprayed withfungal inoculum then placed in humidity chambers for a period of timewhich is found to be optimum for development of each disease. Afterincubation, the plants are moved to the greenhouse, symptoms allowed todevelop, and the plants evaluated for disease intensity. Table 3 reportsthe percent disease control as the average of two replicates.

TABLE 3 Disease (% control) Example Pi Pv As Bc Sn Pr Eg 25  8phytotoxic 5 12 0 0 0 26  0 0 17   3 0 0 0 27 40 0 3  7 0 0 0 28 65phytotoxic 8 33 0 0 0 29 33 0 12  12 0 0 0 Pi. Phyophthora infestans;Late blight tomato Pv: Plasmopara viticola; Grape mildew As: Alternariasolani; Early blight tomato Bc: Botrytis cinerea; Gray mold Sn: Septorianodorum Pr: Puccinia recondita; Wheat leaf rust Eh: Erysiphe graminis;Powdery mildew wheat

Following review of the control achieved by examples 25-29, theconcentration of pentacyclic triterpene and carrier vehicle werechanged. Table 4 identifies the compositions of examples 30-35, andTable 5 shows the percent control of plant diseases following aprotective application of materials in examples 30-35. The results intable 5 are the mean of three replicates.

TABLE 4 Example Composition 30 Carrier vehicle containing 0.35% solutionof Tween 80 ™ in water. 31 200 mg betulin in 100 ml of carrier. 32 400mg betulin in 100 ml of carrier. 33 800 mg betulin in 100 ml of carrier.34 400 mg ursolic acid in 100 ml of carrier.

TABLE 5 Disease (% control) Ex. Pi Pv As Bc Sn Pr Eg Sm-p Sm-t 30 7 0 4394 0 0 0 50 30 31 10  0 47 100  0 0 0 60 20 32 7 37  53 82 0 0 0 65 3033 60  0 63 97 0 0 0 50 30 34 0 0  0 60 0 0 0 40 60 Pi: Phytophthorainfestans; Late blight tomato Pv: Plasmorara viticola; Grape mildew As:Alternaria solani; Early blight tomato Bc: Botrytis cinerea; Gray moldSn: Septoria nodorum Pr: Puccinia recondita; Wheat leaf rust Eh:Erysiphe graminis; Powdery mildew wheat Sm-p: Sclerotinia minor;Sclerotinia blight (on peanut) Sm-t: Sclerotinia minor; Sclerotiniablight (on tomato)

As seen from Table 5, an increase in the amount of applied betulingenerally increased control in susceptible fungus.

What is claimed is:
 1. A composition useful for inhibiting enzyme-basedattacks on plant surfaces comprising: a) a carrier selected from thegroup consisting of aqueous film-forming solutions, surfactants,emulsion forming additives, a polymer, dimethylsulfoxide, ethyl acetate,acetone, methyl ethyl ketone, a polyethylene glycol ester of analiphatic acid, methylene chloride, chloroform, and mixtures thereof,and b) an agent selected from the group consisting of a dye, a foliarfertilizer or insecticide. and c) pentacyclic triterpene compoundexhibiting a structure of formula II or formula III:

wherein: R¹=CH₂O—X—Y², CH₂NHY¹, CH₂NY¹ ₂, CH₂Y³, CH₂NH—X—OH, CH₂NH—X—Y²,CH₂NH—X—Y³, CH₂NH—X—OY¹, CH₂O—X—OY¹, CO₂Y¹, COY³, COY², CHO,CH═N(CH₂)_(m)(O(CH₂)_(m))_(n)R⁴, or CH═N(CH₂)_(m)(O(CH₂)_(m))_(n)Y²; R²,R³=H, OH, OY¹, O—X—OH, O—X—OY¹, O—X—Y², Y³, NHY¹, NY¹ ₂, Y³, NH—X—OH,NH—X—Y², NH—X—Y³, NH—X—OY¹, NY¹—X—OH, NY¹—X—Y², NY¹—X—Y³, or NY¹—X—OY¹;provided that one of R² or R³ is H or that R² and R³ together denotecarbonyl oxygen; R⁴=H, OH, OY¹, or Y³; Y¹=H, alkyl of 1-30 carbon atoms,straight chain or branched, cycloalkyl of 3-30 carbon atoms, alkanyl of3-30 carbon atoms, oxyalkyl of 4-30 carbon atoms, phenylalkyl of 7-30carbon atoms, or phenoxyalkyl of 7-30 carbon atoms; Y²=NH₂, NHY¹, or NY¹₂; Y³=—(O(CH₂)_(m))_(n)R⁴ or —(O(CH₂)_(m))_(n)Y², where m=2-4 andn=1-230; X=—OC(CH₂)_(p)CO— where p=1-22.
 2. A composition according toclaim 1 further comprising a foliar fertilizer, or insecticide.
 3. Agrain grass having external surfaces covered by a composition accordingto claim
 1. 4. A grain grass according to claim 3 selected from thegroup consisting of rye, wheat, or barley.
 5. A method for protectingplants against fungus attack by a process comprising: applying toexposed plant surfaces a carrier and an effective amount of acomposition containing a pentacyclic triterpene compound exhibiting astructure according to formula II or formula III:

wherein: R¹=CH₂O—X—Y², CH₂NHY¹, CH₂NY¹ ₂, CH₂Y³, CH₂NH—X—OH, CH₂NH—X—Y²,CH₂NH—X—Y³, CH₂NH—X—OY¹, CH₂OC(O)—OY¹, CH₂O—X—OY¹, CO₂Y¹, COY³, COY²,CHO, CH═N(CH₂)_(m)(O(CH₂)_(m))_(n)R⁴, orCH═N(CH₂)_(m)(O(CH₂)_(m))_(n)Y²; R², R³=H, OH, OY¹, O—X—OH, O—X—OY¹,O—X—Y², Y³, NHY¹, NY¹ ₂, Y³, NH—X—OH, NH—X—Y², NH—X—Y³, NH—X—OY¹,NY¹—X—OH, NY¹—X—Y², NY¹—X—Y³, or NY¹—X—OY¹; provided that one of R² orR³ is H or that R² and R³ together denote carbonyl oxygen; R⁴=H, OH,OY¹, or Y³; Y¹=H, alkyl of 1-30 carbon atoms, straight chain orbranched, cycloalkyl of 3-30 carbon atoms, alkanyl of 3-30 carbon atoms,oxyalkyl of 4-30 carbon atoms, phenylalkyl of 7-30 carbon atoms, orphenoxyalkyl of 7-30 carbon atoms; Y²=NH₂, NHY¹, or NY¹ ₂;Y³=—(O(CH₂)_(m))_(n)R⁴ where m=2-4 and n=1-230; X=—OC(CH₂)_(p)CO— wherep=1-22.
 6. A method as in claim 5 wherein the applying step comprisesapplying said composition to surfaces of grain grasses.
 7. A method asin claim 5 wherein said carrier is selected from the group consisting ofan aqueous film-forming solution, a surfactant, an emulsion formingadditive, and a polymer.
 8. A method as in claim 5 wherein said carrieris selected from the group consisting of aqueous film-forming solutions,dimethylsulfoxide, ethyl acetate, acetone, methyl ethyl ketone,methylene chloride, chloroform, and mixtures thereof.
 9. A method as inclaim 5 wherein said composition contains a solvent for said pentacyclictriterpene compound.
 10. A method as in claim 9 wherein said solvent isselected from ethyl acetate, acetone, methyl ethyl ketone, ethanol,propanol, isopropanol, methanol, methylene chloride, chloroform, ortheir mixtures.
 11. A method according to claim 9 wherein said solventcontains 1-25 wt % acetone, about 0-10 wt % dimethylsulfoxide, 0-35%polyethyleneglycol ester of an aliphatic acid, and about 0-25 wt % of asurfactant.
 12. A method as in claim 5 wherein said pentacyclictriterpene compound is applied to said plant surfaces at a ratesufficient to prevent pathogenic infections.
 13. A method as in claim 12wherein said pentacyclic triterpene compound is applied to said plantsurfaces at a rate within the range of 0.1-1000 kg/h.
 14. A method as inclaim 13 wherein said pentacyclic triterpene compound is applied to saidplant surfaces at a rate within the range of 0.1-100 kg/h.
 15. Acomposition according to claim 1 containing water and 7-30 grams of theselected pentacyclic triterpene per gallon of water.
 16. A compositionaccording to claim 1 wherein said carrier comprises a solvent forpentacyclic triterpenes that comprises 1-25 wt% acetone, 0-10 wt% DMSO,0-35 wt% of a polyethyleneglycol ester of an aliphatic acid, and 0-25wt% of a surfactant.
 17. A composition according to claim 1 comprising apentacyclic triterpene that exhibits a structure according to FormulaII.
 18. A composition according to claim 1 comprising a pentacyclictriterpene that exhibits a structure according to Formula III.